3D confocal approaches for large pieces of tissue - All about Microscopy, Confocal Microscopy, Fluorescence microscopy and Other Imaging Techniques

First, apologies if this question has been answered in the archives - I couldn't find anything that matched exactly what I want to know.

I am trying to find a way to image the branchial arches of a midgestation mouse embryo immunofluorescently stained with up to 3 antibodies + DAPI counterstain. Branchial arches are structures on the "neck" of the embryo and are about 400um in the shortest dimension at the time I want to image.

I need to take a 3D look to see the patterns of cells and how they overlap. It needs to be high enough resolution to see colocalisation of antibodies in individual cells. My staining works best at 15-20um.

So, my question:

Is the best approach to take 20 sections at 20um (approximately 400um total), stain them, take a z-stack of each section and then align/register them (with e.g. ImageJ/Fiji)?

I have access to a Leica TCS SP2 confocal laser scanning microscope which can perform z-stacks of approx 80um.

Also, generally does anyone have experience of this kind of experiment and, if so, do you have any tips for getting a nice reconstruction at the end?

Finally, if you know of any good guides or papers out there which have attempted similar things they would be extremely helpful.

Sorry for such a late response, and I hope that by now you have solved this issue.

If not, I hope this helps. Your procedure should work effectively. However, I would like to caution that imaging 400um into the sample with confocal settings will not be optimal. 400um is a bit too deep for true confocal imaging using lasers with visible wavelengths (I have done it, and depending on sample type I have gotten mixed results). If you are trying to image cells, and are using true confocal settings, I would suggest a smaller step size than 20um, as most embryonic cells have a smaller diameter than this.

In order to reconstruct 3D images, I generally use either Metamorph, Image Pro Plus, or Macbiophotonics ImageJ (open source found online for free). These programs all produce similar 3D reconstructions, but I would say that ImageJ is the most convenient/easy to use.